We propose to study the molecular mechanisms of three related molecular processes which alter genome structure in the ciliated protozoan Tetrahymena. These events are 1) chromosome breakage and new telomere formation, 2) Internal DNA deletion or DNA splicing, and 3) ribosomal RNA gene amplification. These processes are highly regulated, occurring reproducibly at specific chromosomal locations during a define period of development. They play important roles in nuclear differentiation, and are crucial for understanding genome stability in this and other eukaryotes. In the past granting period we have completed detailed nucleotide sequence analysis of DNAs involved in these processes, and developed the first DNA transformation method for a ciliate. We have begun to introduce engineered DNAs back into developing nuclei using this method, through which we have found a 15 bp sequence which specifies chromosome breakage sites, polypurine sequences necessary for site specific DNA deletion, and a 42 bp inverted repeats important for the formation of palindromic rDNA during amplification. We will continue this approach, and determine the exact cis-acting sequences required for these and other DNA-altering processes. Specifically, we will determine whether different parts of the 15 bp sequence are responsible for the site specificities of DNA cutting and telomere formation; what other sequences may be required for promoting DNA deletion and specifying its sites; how the inverted repeats work to promote palindrome formation; and what sequences are responsible for regulating ribosomal gene replication and amplification. These results should help us determine specific aspects of their underlying mechanisms. We will further isolate proteins or other macro-molecules which carry out these processes from synchronous developing cells, and set up in vitro reaction systems to determine the steps and components involved. By combining these two approaches we hope to understand the molecular mechanisms of these intriguing genetic processes. They are relevant to our understanding of many human diseases including cancer.